Dopamine promotes cognitive effort by biasing the benefits versus costs of cognitive work.

Stimulants such as methylphenidate are increasingly used for cognitive enhancement but precise mechanisms are unknown. We found that methylphenidate boosts willingness to expend cognitive effort by altering the benefit-to-cost ratio of cognitive work. Willingness to expend effort was greater for participants with higher striatal dopamine synthesis capacity, whereas methylphenidate and sulpiride, a selective D2 receptor antagonist, increased cognitive motivation more for participants with lower synthesis capacity. A sequential sampling model informed by momentary gaze revealed that decisions to expend effort are related to amplification of benefit-versus-cost information attended early in the decision process, whereas the effect of benefits is strengthened with higher synthesis capacity and by methylphenidate. These findings demonstrate that methylphenidate boosts the perceived benefits versus costs of cognitive effort by modulating striatal dopamine signaling.

Epidemiology of Flexor Tendon Injuries of the Hand in a Northern Finnish Population.

BACKGROUND: Flexor tendon injuries cause significant morbidity in working-age population. The epidemiology of these injuries in adult population is not well known. The aim of this study was to describe the epidemiology of flexor tendon injuries in a Northern Finnish population. MATERIAL AND METHODS: Data on flexor tendon injuries, from 2004 to 2010, were retrieved from patient records from four hospitals, which offer surgical repair of the flexor tendon injuries in a well-defined area in Northern Finland. The incidence of flexor tendon injury as well as the gender-specific incidence rates was calculated. Mechanism of injury, concomitant nerve injuries, and re-operations were also recorded. RESULTS: The incidence rate of flexor tendon injury was 7.0/100,000 person-years. The incidence was higher in men and inversely related to age. The most common finger to be affected was the fifth digit. In 37% of injuries also digital nerve was affected. The most common finger to have simultaneous digital nerve injury was the thumb. CONCLUSION: Flexor tendon laceration is a relatively rare injury. It predominantly affects working-aged young males and frequently includes a nerve injury, which requires microsurgical skills from the surgeon performing the repair. This study describes epidemiology of flexor tendon injuries and therefore helps planning the surgical and rehabilitation services needed to address this entity.

Upregulation of the mevalonate pathway by cholesterol depletion abolishes tolerance to N-bisphosphonate induced Vγ9Vδ2 T cell cytotoxicity in PC-3 prostate cancer cells.

Zoledronate (ZOL) inhibits farnesyl pyrophosphate synthase leading to intracellular accumulation of isopentenyl pyrophosphate/triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (IPP/ApppI). Cytotoxic Vγ9Vδ2 T cells have been shown to recognize IPP/ApppI in breast cancer cells. Further, human breast cancer cells have been shown to differ remarkably in their ZOL treatment induced IPP/ApppI production and responses to that. In this communication we analysed the responsiveness of prostate cancer cells PC-3 and DU-145, Caki-2 renal carcinoma cells and U87MG glioblastoma cells to ZOL treatment, and the subsequent activation of Vγ9Vδ2 T-cell cytotoxicity. Of the cell lines tested, PC-3 cells were not susceptible to Vγ9Vδ2 T-cell cytotoxicity due to low activity of the mevalonate pathway and low amount of IPP formed. However, the resistance of PC-3 cells to Vγ9Vδ2 T-cell cytotoxicity could be abrogated by upregulation of the mevalonate pathway through cholesterol depletion.

Neuropeptide Y in the noradrenergic neurones induces obesity and inhibits sympathetic tone in mice.

AIM: Neuropeptide Y (NPY) co-localized with noradrenaline in central and sympathetic nervous systems seems to play a role in the control of energy metabolism. In this study, the aim was to elucidate the effects and pathophysiological mechanisms of increased NPY in catecholaminergic neurones on accumulation of body adiposity. METHODS: Transgenic mice overexpressing NPY under the dopamine-beta-hydroxylase promoter (OE-NPY(DßH) ) and wild-type control mice were followed for body weight gain and body fat content. Food intake, energy expenditure, physical activity, body temperature, serum lipid content and markers of glucose homoeostasis were monitored. Thermogenic and lipolytic responses in adipose tissues, and urine catecholamine and tissue catecholamine synthesizing enzyme levels were analysed as indices of sympathetic tone. RESULTS: Homozygous OE-NPY(DßH) mice showed significant obesity accompanied with impaired glucose tolerance and insulin resistance. Increased adiposity was explained by neither increased food intake or fat absorption nor by decreased total energy expenditure or physical activity. Adipocyte hypertrophy and decreased circulating lipid levels suggested decreased lipolysis and increased lipid uptake. Brown adipose tissue thermogenic capacity was decreased and brown adipocytes filled with lipids. Enhanced response to adrenergic stimuli, downregulation of catecholamine synthesizing enzyme expressions in the brainstem and lower adrenaline excretion supported the notion of low basal catecholaminergic activity. CONCLUSION: Increased NPY in catecholaminergic neurones induces obesity that seems to be a result of preferential fat storage. These results support the role of NPY as a direct effector in peripheral tissues and an inhibitor of sympathetic activity in the pathogenesis of obesity.

Suppression of breast cancer cell growth by a monoclonal antibody targeting cleavable ErbB4 isoforms.

ErbB4 isoforms mediate different cellular activities depending on their susceptibility to proteolytic cleavage. The biological significance of ErbB4 cleavage in tumorigenesis, however, remains poorly understood. Here, we describe characterization of a monoclonal antibody (mAb 1479) that selectively recognizes the ectodomain of cleavable ErbB4 JM-a isoforms both in vitro and in vivo. mAb 1479 was used to analyse ErbB4 JM-a expression and ectodomain shedding in a series of 17 matched breast cancer/histologically normal peripheral breast tissue pairs. ErbB4 ectodomain was observed in 75% of tumors expressing ErbB4 but only in 18% of normal breast tissue samples expressing ErbB4. Difference in the relative quantity of ErbB4 ectodomain between normal and tumor tissue pairs was statistically significant (P=0.015). Treatment with mAb 1479 suppressed ErbB4 function by inhibiting ErbB4 tyrosine phosphorylation and ectodomain shedding, and by stimulating ErbB4 downregulation and ubiquitination. mAb 1479 suppressed both anchorage-dependent and -independent growth of human breast cancer cell lines that naturally express cleavable ErbB4 JM-a. These findings indicate that ErbB4 ectodomain shedding is enhanced in breast cancer tissue in vivo, and that mAb 1479 represents a potential drug candidate that suppresses breast cancer cell growth by selectively binding cleavable ErbB4 isoforms.

Differential nuclear localization and kinase activity of alternative ErbB4 intracellular domains.

Cleavable isoforms of the ErbB4 receptor tyrosine kinase release a soluble intracellular domain (ICD) that may translocate to the nucleus and regulate signaling. However, ErbB4 gene is alternatively spliced generating CYT-1 and CYT-2 isoforms with different cytoplasmic tails. Here, we addressed whether the two alternative ErbB4 ICDs of either CYT-1 (ICD1) or CYT-2 (ICD2) type differ in signaling to the nucleus. Confocal microscopy and extraction of nuclear cell fractions indicated that significantly more ICD2 translocated to the nuclei when compared to ICD1. Unlike the membrane-anchored 80 kDa fragments derived from full-length ErbB4 isoforms, the two ICDs did not differ from each other in metabolic stability or ubiquitylation. However, ICD2 was phosphorylated at tyrosine residues to a higher extent and demonstrated greater in vitro kinase activity than ICD1. Mutating the ATP-binding site within ICD2 kinase domain (ICD2 K751R) blocked its tyrosine phosphorylation and significantly reduced its nuclear translocation. When expressed in the context of full-length ErbB4, ICD2 was also more efficient than ICD1 in promoting transcriptional activation of the STAT5 target gene beta-casein. These findings indicate that the two alternative ICDs of ErbB4 differ in their nuclear accumulation, and that the mechanism involves differential kinase activity but not ubiquitin-regulated ICD stability.

Inhibition of the membrane fusion machinery prevents exit from the TGN and proteolytic processing by furin.

The Semliki Forest virus (SFV) glycoprotein precursor p62 is processed to the E2 and E3 during the transport from the trans-Golgi network (TGN) to the cell surface. We have studied the regulation of the membrane fusion machinery (Rab/N-ethylmaleimide (NEM)-sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)-SNAP receptor) in this processing. Activation of the disassembly of this complex with recombinant NSF stimulated the cleavage of p62 in permeabilized cells. Inactivation of NSF with a mutant alpha-SNAP(L294A) or NEM treatment inhibited processing of p62. Rab GDP dissociation inhibitor inhibited the cleavage. Inactivation of NSF blocks the transport of SFV glycoproteins and vesicular stomatitis virus G-glycoprotein from the TGN membranes to the cell surface. The results support the conclusion that inhibition of membrane fusion arrests p62 in the TGN and prevents its processing by furin.

Limited caspase cleavage of human BAP31.

Human BAP31 was cleaved at both of its two identical caspase cleavage sites in two previously reported models of apoptosis. We show here that only the most carboxy-terminal site is cleaved during apoptosis induced in HeLa cells by tunicamycin, tumor necrosis factor and cycloheximide, or staurosporine. Similar results were obtained in HL-60 cells using Fas/APO-1 antibodies, or cycloheximide. This limited cleavage, which is inhibited by several caspase inhibitors, removes eight amino acids from human BAP31 including the KKXX coat protein I binding motif. Ectopic expression of the resulting cleavage product induces redistribution of mannosidase II from the Golgi and prevents endoplasmic reticulum to Golgi transport of virus glycoproteins.

Chemokine expression by central nervous system resident cells and infiltrating neutrophils during experimental autoimmune encephalomyelitis in the BALB/c mouse.

The active role of chemokines in the central nervous system (CNS) during the pathogenesis of experimental autoimmune encephalomyelitis (EAE) has been clearly established. In this study the expression pattern of several chemokines and cytokines was elucidated using reverse transcription-PCR and immunohistochemistry in a recently established EAE model of the BALB/c mouse that is characterized by CNS infiltration of polymorphonuclear neutrophils. Elevated mRNA levels of the chemokines MIP-1alpha, MIP-2 and MCP-1 were detected in the CNS of diseased mice, whereas no chemokine expression could be measured in asymptomatic mice. Activated astrocytes were shown to be the main source of MIP-1alpha and MIP-2 before and during cellular CNS infiltration. Among the infiltrating immune cells the neutrophils secreted MIP-1alpha and MCP-1. These results suggest involvement of ordered chemokine expression during the process of neutrophil attraction into the CNS, which may play an important role in the initiation and perpetuation of autoimmune CNS inflammation in the BALB/c mouse. This is the first EAE model to describe CNS expression of the C-X-C chemokine MIP-2, corresponding to an observed neutrophil accumulation in the CNS.

Replicase complex genes of Semliki Forest virus confer lethal neurovirulence.

Semliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with SFV4, a neurovirulent strain of SFV, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent A7(74) strain remain asymptomatic. In adult neurons, A7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. To elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotide genome of A7(74) and compared it to the highly neurovirulent SFV4 virus. We found several sequence differences and sought to localize determinants conferring the neuropathogenicity by using a panel of chimeras between SFV4 and a cloned recombinant, rA774. We first localized virulence determinants in the nonstructural region by showing that rA774 structural genes combined with the SFV4 nonstructural genome produced a highly virulent virus, while a reciprocal recombinant was asymptomatic. In addition to several amino acid mutations in the nonstructural region, the nsp3 gene of rA774 displayed an opal termination codon and an in-frame 21-nucleotide deletion close to the nsp4 junction. Replacement in rA774 of the entire nsp3 gene with that of SFV4 reconstituted the virulent phenotype, whereas an arginine at the opal position significantly increased virulence, leading to clinical symptoms in mice. Completion of the nsp3 deletion in rA774 did not increase virulence. We conclude that the opal codon and amino acid mutations other than the deleted residues are mainly responsible for the attenuation of A7(74) and that the attenuating determinants reside entirely in the nonstructural region.

A natural ErbB4 isoform that does not activate phosphoinositide 3-kinase mediates proliferation but not survival or chemotaxis.

ErbB4 is a member of the epidermal growth factor receptor (ErbB) family that mediates cellular responses activated by neuregulins (NRG) and other epidermal growth factor-like growth factors. Two naturally occurring ErbB4 isoforms, ErbB4 CYT-1 and ErbB4 CYT-2, have previously been identified. Unlike ErbB4 CYT-1, ErbB4 CYT-2 lacks a phosphoinositide 3-kinase (PI3-K)-binding site and is incapable of activating PI3-K. We have now examined the consequences of the inability of this isoform to activate PI3-K on cell proliferation, survival, and chemotaxis in response to NRG-1beta: (i) NRG-1beta stimulated proliferation of cells expressing either ErbB4 CYT-1 or ErbB4 CYT-2. Consistent with the mitogenic responsiveness, analysis of downstream signaling showed that Shc and MAPK were phosphorylated after stimulating either isoform with NRG-1beta. (ii) NRG-1beta protected cells expressing ErbB4 CYT-1 but not cells expressing ErbB4 CYT-2 from starvation-induced apoptosis as measured by effects on cell number and 4', 6-diamidino-2-phenylindole staining. Furthermore, in cells expressing ErbB4 CYT-2, Akt, a protein kinase that mediates cell survival, was not phosphorylated. (iii) NRG-1beta stimulated chemotaxis and membrane ruffling in cells expressing ErbB4 CYT-1 but not in cells expressing ErbB4 CYT-2. In summary, ErbB4 CYT-2 can mediate proliferation but not chemotaxis or survival. These results suggest a novel mechanism by which cellular responses such as chemotaxis and survival may be regulated by the expression of alternative receptor-tyrosine kinase isoforms that differ in their coupling to PI3-K signaling.

Mobilization of late-endosomal cholesterol is inhibited by Rab guanine nucleotide dissociation inhibitor.

Cholesterol entering cells in low-density lipoproteins (LDL) via receptor-mediated endocytosis is transported to organelles of the late endocytic pathway for degradation of the lipoprotein particles. The fate of the free cholesterol released remains poorly understood, however. Recent observations suggest that late-endosomal cholesterol sequestration is regulated by the dynamics of lysobisphosphatidic acid (LBPA)-rich membranes [1]. Genetic studies have pinpointed a protein, Niemann-Pick C-1 (NPC-1), that is required for the mobilization of late-endosomal/lysosomal cholesterol by an unknown mechanism [2]. Here, we report the removal of accumulated cholesterol by overexpression of the NPC-1 protein in NPC-1-deficient fibroblasts from patients with Niemann-Pick disease, and in normal fibroblasts upon release of a progesterone-induced block of cholesterol transport. We show that late-endosomal/lysosomal cholesterol mobilization is specifically inhibited by microinjection of Rab GDP-dissociation inhibitor (Rab-GDI). Moreover, clearance of the cholesterol deposits by NPC-1 in patients' fibroblasts is accompanied by the redistribution of LBPA and of a lysosomal hydrolase that utilizes the mannose-6-phosphate receptor. Our results reveal, for the first time, the involvement of a specific molecular component of the membrane-trafficking machinery in cholesterol transport and the coupling of late-endosomal cholesterol egress to the trafficking of other lipid and protein cargo.

Enhancement of experimental autoimmune encephalomyelitis severity by ultrasound emulsification of antigen/adjuvant in distinct strains of mice.

Susceptibility to experimental autoimmune encephalomyelitis (EAE) is associated with the major histocompatibility complex (MHC) haplotype. In this study EAE could be induced in six out of ten mice of the resistant DBA/2 (H-2d) strain by ultrasound emulsified antigen/adjuvant, whereas none of the mice immunized with the conventional adjuvant developed the disease. Similar results were previously obtained for the MHC identical BALB/c mice. Further, while only few T cells were present in the central nervous systems (CNS) of the diseased DBA/2 mice, macrophages formed the majority of the infiltrates. In congenic BALB.B (H-2b) and BALB.K (H-2 k) mice, EAE could be induced with both sonicated and extruded antigen/adjuvant emulsion. The results indicate that the EAE resistance in mice carrying the H-2d MHC haplotype is dependent on the physical structure of the immunogen.

Erbb4 and its isoforms: selective regulation of growth factor responses by naturally occurring receptor variants.

ErbB4 is a member of the epidermal growth factor receptor (EGFR, ErbB) family that mediates responses to neuregulins and other EGF-like growth factors. ErbB4 is a central regulator of cardiovascular and neural development as well as differentiation of the mammary gland. A role for ErbB4 has also been implicated in malignancies and heart diseases. Four structurally and functionally distinct ErbB4 isoforms have recently been identified. One pair of isoforms differs within their extracellular juxtamembrane domains. These juxtamembrane ErbB4 isoforms are either susceptible or resistant to proteolytic processing that release a soluble receptor ectodomain. Another pair of ErbB4 isoforms differs within their cytoplasmic tails. Analysis of the intracellular signal transduction pathways indicates that both cytoplasmic ErbB4 isoforms can couple to the Shc-MAPK signaling pathway, while the other one is incapable of coupling to the phosphoinositide 3-kinase (PI3-K)-Akt pathway. The differences in the activation of signaling cascades are reflected in the cellular responses stimulated via the cytoplasmic isoforms. Both cytoplasmic ErbB4 isoforms can stimulate proliferation, but the isoform that cannot activate PI3-K is defective in stimulating cellular survival and chemotaxis. Together these four naturally occurring receptor variants provide a new level of diversity to the control of growth factor-stimulated cellular responses. Thus, the ErbB4 isoforms may have distinct and specific roles in the regulation of various developmental and pathological processes.

Neutrophils secreting tumor necrosis factor alpha infiltrate the central nervous system of BALB/c mice with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) can be induced in resistant BALB/c mice by ultrasound-formed adjuvant emulsion. In contrast to susceptible mouse strains large numbers of neutrophils secreting TNF-alpha occupied the central nervous system (CNS) of BALB/c mice with severe EAE, whereas only small numbers of macrophages and CD4+ T-cells could be detected. CNS infiltration was preceded with activation of microglial cells. Ultrasound formed adjuvant induced early IFN-gamma expression in popliteal lymph nodes of BALB/c mice, whereas conventional adjuvant induced delayed IFN-gamma production. Although the clinical outcome of EAE was similar to that seen in susceptible mice, the pathogenesis was distinct having possible implications on the different forms seen in multiple sclerosis.

Encephalitogenicity of myelin-associated oligodendrocytic basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase for BALB/c and SJL mice.

In search of new encephalitogenic myelin antigens, the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and 19 000 MW isoform of myelin-associated oligodendrocytic basic protein (MOBP) were obtained as recombinant proteins by the baculovirus expression system in Spodoptera frugiperda cells and purified to homogeneity by immobilized metal chelate affinity chromatography (IMAC). The purified MOBP was soluble in water and showed retarded migration on sodium dodecyl sulphate-polyacrylamide gel electrophoresis similar to myelin basic protein (MBP). MOBP induced experimental autoimmune encephalomyelitis (EAE) in nine of 15 susceptible SJL OlaHsd mice, causing death in two animals, whereas three of 14 BALB/c mice showed mild symptoms of EAE, manifested as transient weakness of hind limbs. In both mouse strains, periventricular infiltrates of mononuclear cells were observed. In addition, both 46 000 MW and 48 000 MW CNP isoforms were shown to be non-encephalitogenic for both mouse strains.

The significance of the 3'-nontranslated region and E2 amino acid mutations in the virulence of Semliki Forest virus in mice.

We have recently shown that the 3'-nontranslated region (3'-NTR) of the avirulent Semliki Forest virus A7(74) [SFVA7(74)] contains a unique sequence of 101 nucleotides and five repetitive nucleotide units whereas the 3'-NTR of the neurovirulent SFV4 has only two repeats. A chimeric virus was constructed by replacing the entire 3'-NTR of the SFV4 clone with the A7(74) 3'-NTR. The hybrid replicated efficiently in the central nervous system (CNS) of adult Balb/c mice and, similarly to SFV4, led to high mortality after intraperitoneal inoculation. In contrast, another chimeric virus, CME2, containing the E2 gene of the avirulent SFVA7(74) virus in the SFV4 clone was recently shown to be avirulent for mice. Several derivatives with single-site or a constellation of amino acid mutations were constructed. Two single-site E2 mutants, Val37lle and Asn212Ser, displayed an attenuated phenotype in mice with mortality reduced from 90 to 48 and 43%, respectively. None of the multiple site mutants were significantly attenuated. Adult female mice showed a greater resistance to SFV infection than male mice. The SFV hybrid viruses, CM3NTR and CME2, reached the CNS similarly to the parental viruses, but the single-site E2 mutants were only sporadically found in the CNS. We conclude that in mice the 3'-NTR does not play a significant role in the pathogenesis of Semliki Forest virus and that specific E2 amino acid mutations reduce the virulence, especially in female mice. The results additionally suggest that individual amino acid mutations in the E2 glycoprotein affect the efficiency of migration into the CNS.

Detection of myelin basic protein isoforms by organic concentration.

An effective technique was developed, which allowed rapid isolation of highly pure myelin basic protein (MBP) including its distinct isoforms. The procedure employs homogenization of central nervous system (CNS) tissue in chloroform, which specifically extracts MBP. Subsequently, methanol was used to convert the protein susceptible to quantitative transfer into the acidic aqueous phase. MBP was purified from bovine, chicken, fish, human, guinea-pig, mouse, rabbit, rat, and swine brains. Analysis on SDS-PAGE and immunoblotting using polyclonal MBP-specific serum recognized proteins corresponding to the sizes of previously identified MBP isoforms of 21.5, 18.5, 17.2, and 14.2 kDa and three predicted isoforms of 20.2, 16.0, and 13 kDa. The MBP obtained was readily soluble in water and possessed the capacity to induce experimental autoimmune encephalomyelitis in susceptible mice. The protein was also suitable for use as a substrate for protein kinases.

Physical state of the neuroantigen in adjuvant emulsions determines encephalitogenic status in the BALB/c mouse.

A novel form of adjuvant-neuroantigen formulation was established which was highly encephalitogenic in previously resistant BALB/c mice. The antigen formulation contained mouse whole spinal cord homogenate (MSCH), mycobacteria, and mineral oil, identically to the conventional preparation, but emulsification was completed by sonication instead of extrusion. Sonication of MSCH alone did not render a conventionally prepared emulsion encephalitogenic. The novel adjuvant formulation showed reduced water-oil droplet size, and the neuroantigen was located on the surface of the droplets as well as in the intermicellar space, while in the extruded formulation the material was buried in the droplet interior. Mice inoculated with the sonicated emulsion showed strong brain and spinal cord infiltration of lymphoid cells. The sonicated emulsion was highly encephalitogenic in all six BALB/c substrains tested. The results suggest that availability of the neuroantigen is of critical importance for the development of clinical EAE in the BALB/c mouse.

The Semliki Forest virus E2 gene as a virulence determinant.

We have determined the nucleotide sequences of the capsid, E3, E2 and 6K genes of the avirulent Semliki Forest virus variant A774 (SFV A7). The sequence analysis revealed a nucleotide identity of 98% for capsid, 98% for E3, 97% for E2 and 98% for 6K genes, as compared with the prototype SFV strain L10. At the protein level, the capsid and E3 polypeptides of SFV A7 both exhibited two amino acid substitutions, whereas point mutations in the 6K gene did not alter the amino acid sequence. In the E2 gene of SFV A7, seven of the 34 point mutations led to an amino acid difference as compared with the L10 strain. Replacement of the E2 glycoprotein gene of the virulent SFV4 clone with the corresponding region of SFV A7 resulted in a new plasmid construct, pME2, that gave rise to infectious virus CME2. CME2 and SFV4 replicated similarly in an immortalized mouse brain cell line (MBA 13). Intraperitoneal injection of 10(6) p.f.u. of CME2 into 4- to 6-week-old BALB/c mice caused mild clinical signs in some mice, whereas the majority of the infected animals remained asymptomatic, similar to infection with the avirulent SFV A7. In contrast, infection with the parental SFV4, a derivative of the virulent L10 strain, was lethal in 80% of mice. Virus titres in blood and brain tissue specimens of BALB/c mice were similar after infection with CME2 or A7 viruses. The results suggest that amino acid differences in the E2 glycoprotein individually or in concert cause the attenuation of CME2.